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Nature carefully selects specific metal ions for incorporation into the enzymes that catalyse the chemical reactions necessary for life. Hydrogenases, enzymes that activate molecular H2, exclusively utilize Ni and Fe in [NiFe]-, [FeFe]- and [Fe]-hydrogeanses. However, other transition metals are known to activate or catalyse the production of hydrogen in synthetic systems. Here, we report the development of a biomimetic model complex of [Fe]-hydrogenase that incorporates a Mn, as opposed to a Fe, metal centre. This Mn complex is able to heterolytically cleave H2as well as catalyse hydrogenation reactions. The incorporation of the model into an apoenzyme of [Fe]-hydrogenase results in a [Mn]-hydrogenase with an enhanced occupancy-normalized activity over an analogous semi-synthetic [Fe]-hydrogenase. These findings demonstrate a non-native metal hydrogenase that shows catalytic functionality and that hydrogenases based on a manganese active site are viable. Natural hydrogenases exclusively utilize Ni and/or Fe to activate or produce hydrogen. Now, a catalytically active [Mn]-hydrogenase has been prepared by incorporating a synthetic Mn complex into the apoenzyme of [Fe]-Hydrogenase. The semi-synthetic [Mn]-hydrogenase shows higher activity than the corresponding Fe analogue.

Metabolism establishes a potential difference across the cell membrane of every living cell which drives and regulates secondary ion and solute transfer across membrane proteins. Unraveling the effect of the membrane potential on the level of single molecular groups of the membrane protein was long hampered by the lack of appropriate analytical techniques. We have developed Surface Enhanced Infrared Difference Absorption Spectroscopy (SEIDAS), a highly sensitive vibrational technique for surface analysis, for the study of solid-supported monolayers of orientated membrane proteins. Here, we present spectroscopic data on vibrational changes of sensory rhodopsin II from Natronomonas pharaonis(NpSR II). The application of the electrode potential provides a voltage drop across the NpSR II monolayer through the Helmholtz double layer that mimics the cellular membrane potential. IR difference spectra indicated a shift of the photostationary equilibrium from an M and O mixture toward an M dominant equilibrium. The shift of positive to negative potential exhibited similar effects on the light-induced SEIDA spectra as the increase in pH. This effect is explained in terms of local pH change raised by the compensation of excess charge from the electrode. As we have shown earlier (Jiang, et al. Proc. Natl. Acad. Sci. U.S.A.2008, 105(34), 12113−12117), the application of an electric field opposite to the physiological proton transfer from the retinal Schiff base to its counterion Asp75 leads to the selective halt of the latter. However, when the solution pH is much higher than 5.8, that is, when the proton releasing group at the extracellular side is ionized, proton transfer of Asp75 becomes insensitive to the electric field exerted by the electrode. We infer that the deprotonation of the proton release group creates a local polar environment surrounding Asp75 as a consequence of hydrogen-bonding rearrangements that exceeds the energy of the external dipole. Our results reveal a molecular model for the physiological regulation of the photocycle of NpSR II by the potential drop across the membrane which came about by the interplay between the change in local pH at the membrane surface and the external electric field.

Carbapenem resistance mediated by metallo-β-lactamases (MBL) such as New Delhi metallo-β-lactamase-1 (NDM-1) has become a major factor threatening the efficacy of essential β-lactam antibiotics. Starting from hit fragment dipicolinic acid (DPA), 8-hydroxy- and 8-sulfonamido-quinoline-2-carboxylic acids were developed as inhibitors of NDM-1 with highly improved inhibitory activity and binding affinity. The most active compounds formed reversibly inactive ternary protein-inhibitor complexes with two zinc ions as proven by native protein mass spectrometry and bio-layer interferometry. Modification of the NDM-1 structure with remarkable entropic gain was shown by isothermal titration calorimetry and NMR spectroscopy of isotopically labeled protein. The best compounds were potent inhibitors of NDM-1 and other representative MBL with no or little inhibition of human zinc-binding enzymes. These inhibitors significantly reduced the minimum inhibitory concentrations (MIC) of meropenem for multidrug-resistant bacteria recombinantly expressing blaNDM-1as well as for several multidrug-resistant clinical strains at concentrations non-toxic to human cells.

Hydrogenases are iron–sulfur proteins that catalyze hydrogen turnover in a wide range of microorganisms. Three different classes have been described, and among these [FeFe] hydrogenases are the most active in H2evolution. Hydrogenases are redox enzymes that have been shown to exchange electrons with graphite and modified noble metal electrodes. Making use of the latter, diffusible electron carriers are required to enable redox catalysis, as proteins do not specifically bind to the electrode surface. Diffusion‐limited electron transfer can be replaced by electron injection into immobilized hydrogenase by binding the redox mediator to the electrode surface. Here, we present the synthesis and spectroelectrochemical characterization of 1‐(10‐mercaptodecyl)‐1′‐benzyl‐4,4′‐bipyridinium dibromide (MBBP), which is used as redox‐active linker. CrHydA1, the high‐activity [FeFe] hydrogenase from Chlamydomonas reinhardtii, is immobilized on the linker‐modified gold electrode. Each surface modification step is controlled in situ by surface‐enhanced infrared absorption spectroscopy (SEIRAS). Functionality of the electrode–protein hybrid is demonstrated by recording the linker‐supported current. The specificcatalytic rate of hydrogen evolution by CrHydA1 (2.9 μmol H2min–1mg–1hydrogenase) promises a valuable approach for further optimization of this novel bioelectrical interface.

New techniques in vibrational spectroscopy are promising for the study of biological samples as they provide exquisite spatial andor temporal resolution with the benefit of minimal perturbation of the system during observation. In this Minireview we showcase the power of modern infrared techniques when applied to biological and biomimetic systems. Examples will be presented on how conformational changes in peptides can be traced with femtosecond resolution and nanometer sensitivity by 2D IR spectroscopy, and how surfaceenhanced infrared difference absorption spectroscopy can be used to monitor the effect of the membrane potential on a single protontransfer step in an integral membrane protein. Vibrational spectra of monolayers of molecules at basically any interface can be recorded with sumfrequency generation, which is strictly surfacesensitive. Chemical images are recorded by applying scanning nearfield infrared microscopy at lateral resolutions better than 50nm.

Neue schwingungsspektroskopische Techniken sind sehr vielversprechende Methoden zur Untersuchung biologischer Materialien, da sie eine sehr hohe räumliche und zeitliche Auflösung bei gleichzeitiger minimaler Einflussnahme auf das zu untersuchende System ermöglichen. In diesem Kurzaufsatz werden diese Techniken und ihre potenzielle Bedeutung für biologische und biomimetische Systeme diskutiert. Vorgestellt werden z. B. die Verfolgung von Konformationsänderungen in Peptiden mit fs‐Auflösung und nm‐Empfindlichkeit mit 2D‐IR‐Spektroskopie sowie die Untersuchung des Effekts eines angelegten Membranpotentials auf einzelne Protonentransferschritte innerhalb integraler Membranproteine mit oberflächenverstärkter Infrarot‐Differenz‐Absorptionsspektroskopie. Schwingungsspektren einer molekularen Monoschicht können mithilfe der Summenfrequenz‐Erzeugung registriert werden, während die Rasternahfeld‐IR‐Mikroskopie die Aufnahme chemischer Abbilder einer Oberfläche mit einer lateralen Auflösung < 50 nm ermöglicht.

Nature provides key components for generating fuels from renewable resources in the form of enzymatic nanomachines which catalyze crucial steps in biological energy conversion, for example, the photosynthetic apparatus, which transforms solar power into chemical energy, and hydrogenases, capable of generating molecular hydrogen. As sunlight is usually used to synthesize carbohydrates, direct generation of hydrogen from light represents an exception in nature. On the molecular level, the crucial step for conversion of solar energy into H2lies in the efficient electronic coupling of photosystem I and hydrogenase. Here we show the stepwise assembly of a hybrid complex consisting of photosystem I and hydrogenase on a solid gold surface. This device gave rise to light-induced H2evolution. Hydrogen production is possible at far higher potential and thus lower energy compared to those of previously described (bio)nanoelectronic devices that did not employ the photosynthesis apparatus. The successful demonstration of efficient solar-to-hydrogen conversion may serve as a blueprint for the establishment of this system in a living organism with the paramount advantage of self-replication.

Surface-enhanced infrared adsorption spectroscopy (SEIRAS) was employed for the in situ observation of structural changes that occur in a self-assembled monolayer (SAM) of 11-mercaptoundecanoic acid (MUA) bound on a gold surface. The observed SEIRA spectra reveal a deprotonation of the carboxyl head group of the MUA-SAM layer after adsorption. An analysis of the vibrational spectra suggests that the deprotonation process occurs when the adsorbed MUA molecules reach a critical mutual distance. MUA-SAMs promote direct electron transfer between the metal electrode and cytochrome c, the electron mediator between the integral membrane protein complexes of the respiratory chain. The results show that the coverage of cytochrome c increases with the coverage of deprotonated MUA on the surface. On the other hand, the electron transfer of cytochrome c is optimized only when a moderate amount of the carboxyl head group is deprotonated. The electron transfer of cytochrome c is suppressed with a further increase of the deprotonated MUA. The relationship between the surface structure of the MUA layer and the electron transfer of cytochrome c is discussed on the basis of the spectroscopic data.

Cytochrome coxidase was tethered to a functionalised Ag electrode viaa histidine-tag on the C-terminus of subunit I or II and embedded in a phospholipid bilayer. The uniformly oriented membrane-bound proteins were studied by surface enhanced resonance Raman spectroscopy (SERRS) that reveals preservation of the native structures of the heme aand heme a3sites. On the basis of time-dependent SERRS measurements, the rate constant for the heterogeneous electron transfer to heme awas determined to be 0.002 s−1independent of the enzyme orientation and the overpotential. Taking into account that the electrode-to-heme adistance is larger than 50 , these findings suggest an electron hopping mechanism in which the CuAcenter is not involved. Electrochemical reduction is restricted to heme awhereas electron transfer from heme ato heme a3, which in solution occurs on the nanosecond time scale, is drastically slowed down. It may be that the network of cooperativities that links intramolecular electron transfer and proton translocation is perturbed in the immobilised enzyme, possibly due to the effect of the interfacial electric field.

The interplay of fluorination and structure of alkane self-assembled monolayers and how these affect hydrophobicity are explored via molecular dynamics simulations, contact angle goniometry, and surface-enhanced infrared absorption spectroscopy. Wetting coefficients are found to grow linearly in the monolayer density for both alkane and perfluoroalkane monolayers. The larger contact angles of monolayers of perfluorinated alkanes are shown to be primarily caused by their larger molecular volume, which leads to a larger nearest-neighbor grafting distance and smaller tilt angle. Increasing the Lennard-Jones force cutoff in simulations is found to increase hydrophilicity. Specifically, wetting coefficients scale like the inverse square of the cutoff, and when extrapolated to the infinite cutoff limit, they yield contact angles that compare favorably to experimental values. Nanoscale roughness is also found to reliably increase monolayer hydrophobicity, mostly via the reduction of the entropic part of the work of adhesion. Analysis of depletion lengths shows that droplets on nanorough surfaces partially penetrate the surface, intermediate between Wenzel and Cassie–Baxter states.

Membrane-bound cytochrome coxidase was attached to an electrode viaa His-tag linker and studied by surface enhanced resonance Raman spectroscopy, demonstrating intact redox site structures and electron transfer between the electrode and the immobilized enzyme.

Membrane-bound cytochrome c oxidase was attached to an electrode via a His-tag linker and studied by surface enhanced resonance Raman spectroscopy, demonstrating intact redox site structures and electron transfer between the electrode and the immobilized enzyme.

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